Thienopyrimidine compounds, their production and use

ABSTRACT

A compound of formula (I) wherein R 1  and R 2  each is hydrogen, hydroxy, C 1-4  alkoxy, C 1-4  alkoxy-carbonyl or C 1-4  alkyl which may be substituted; R 3  is hydrogen, halogen, hydroxy or C 1-4  alkoxy which may be substituted; or adjacent two R 3  may form C 1-4  alkylenedioxy; R 4  is hydrogen or C 1-4  alkyl; R 6  is C 1-4  alkyl which may be substituted or a group of the formula (A) wherein R 5  is hydrogen of R 4  and R 5  may form heterocycle; and n is 0-5, or a salt thereof, has an excellent GnRH-antagonizing activity, and is useful for preventing or treating sex hormone-dependent diseases.

This application is a 371 of PCT/JP00/01777 filed Mar. 23, 2000.

TECHNICAL FIELD

The present invention relates to thieno[2,3-d]pyrimidine compoundsexhibiting gonadotropin releasing hormone (GnRH) antagonizing activity,their production and use.

BACKGROUND ART

Secretion of anterior pituitary hormones undergoes feedback control byperipheral hormones secreted from target organs of the respectivehormones and by secretion-regulating hormones from the hypothalamus,which is the upper central organ of the anterior lobe of the pituitary(hereinafter, these hormones are collectively called “hypothalamichormones” in this specification). At the present stage, for hypothalamichormones, the existence of nine kinds of hormones including, forexample, thyrotropin releasing hormone (TRH), and gonadotropin releasinghormone [GnRH, sometimes called LH-RH (luteinizing hormone releasinghormone)] has been confirmed. These hypothalamic hormones are believedto show their actions via the receptors which are considered to exist inthe anterior lobe of the pituitary, and efforts to find thereceptor-gene expression specific to these hormones, including forhumans, have been made. Accordingly, antagonists or agonistsspecifically and selectively acting on these receptors should controlthe action of the hypothalamic hormone and the secretion of anteriorpituitary hormone. As a result, such antagonists or agonists areexpected to be useful in preventing treating anterior pituitary hormonediseases.

Known compounds possessing GnRH-antagonizing activity includeGnRH-derived linear peptides (U.S. Pat. No. 5,140,009 and U.S. Pat. No.5,171,835), a cyclic hexapeptide derivative (JP-A-61-191698), a bicyclicpeptide derivative [Journal of Medicinal Chemistry, Vol. 36, pp.3265-3273 (1993)], and so forth. Non-peptide compounds possessingGnRH-antagonizing activity include compounds described in WO 95/28405(JP-A-8-295693), WO 97/14697 (JP-A-9-169767), WO 97/14682(JP-A-9-169735) and WO 96/24597 (JP-A-9-169768), etc.

Peptide compounds pose a large number of problems to be resolved withrespect to oral absorbability, dosage form, dose volume, drug stabilitysustained action, metabolic stability etc. There is strong demand for anoral GnRH antagonist, especially one based on a non-peptide compound,that has excellent therapeutic effect on hormone-dependent cancers,e.g., prostatic cancer, endometriosis, precocious puberty etc., thatdoes not show transient hypophysial-gonadotropic action (acute action)and that has excellent bioavailability.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the percent LH concentrations in test monkey plasma. In thefigure, -▴- represents control group-1, -♦- represents control group-2,-Δ- represents compound group-1, -□- represents compound group-2 and -◯-represents compound group-3, respectively.

DISCLOSURE OF INVENTION

We, the present inventors, have studied various compounds, and as aresult, have found for the first time the following novel compound whichhas a substituent, a group of the formula: —NH—CO—NR¹R² wherein eachsymbol is as defined below, on the para-position of the phenyl group ofthe thieno[2,3-d]pyrimidine skeleton, or a salt thereof [hereinaftersometimes referred to briefly as compound (I)]. And we also have foundout that compound (I) has an unexpected, excellent GnRH-antagonizingactivity, based upon the above specific substituent, and low toxicityand is therefore satisfactory as a medicine having GnRH-antagonizingactivity, and developed the present invention based on this finding.

wherein R¹ and R² each represents a hydrogen atom, a hydroxy group, aC₁₋₄ alkoxy group, a C₁₋₄ alkoxy-carbonyl group or a C₁₋₄ alkyl groupwhich may be substituted;

R³ represents a hydrogen atom, a halogen atom, a hydroxy group or a C₁₋₄alkoxy group which may be substituted; or adjacent two R³ may form,taken together, a C₁₋₄ alkylenedioxy group;

R⁴ represents a hydrogen atom or a C₁₋₄ alkyl group;

R⁶ represents a C₁₋₄ alkyl group which may be substituted or a group ofthe formula:

 wherein R⁵ represents a hydrogen atom or R⁴ and R⁵ may form, takentogether, a heterocycle; and

n represents an integer of 0 to 5.

Accordingly, the present invention relates to:

[1] a compound (I);

[2] a compound of the above [1] or a salt thereof, which is a compoundof the formula:

 wherein R¹ and R² each is a hydrogen atom, a hydroxy group, a C₁₋₄alkoxy group or a C₁₋₄ alkyl group which may be substituted; R³ is ahydrogen atom, a halogen atom or a C₁₋₄ alkoxy group; R⁴ is a C₁₋₄ alkylgroup; and R⁵ is as defined above;

[3] a compound of the above [1] or a salt thereof, wherein R¹ is a C₁₋₃alkoxy group;

[4] a compound of the above [3] or a salt thereof, wherein R² is ahydrogen atom;

[5] a compound of the above [1] or a salt thereof, wherein R³ is ahydrogen atom;

[6] a compound of the above [1] or a salt thereof, wherein R⁶ is a groupof the formula:

 wherein R⁵ is as defined above;

[7] a compound of the above [2] or a salt thereof, wherein R⁴ is a C₁₋₃alkyl group and R⁵ is a hydrogen atom;

[8] a compound of the above [1] or a salt thereof, wherein n is 1 or 2;

[9] a compound of the above [1] or a salt thereof, wherein R¹ is (i) ahydroxy group, (ii) a C₁₋₄ alkoxy group, or (iii) a C₁₋₄ alkyl groupwhich may be substituted by hydroxy or C₁₋₄ alkyl-carbonyloxy; R² is ahydrogen atom, a C₁₋₄ alkyl group or a C₁₋₄ alkoxy-carbonyl group; R³ isa hydrogen atom, a halogen atom, a hydroxy group or a C₁₋₄ alkoxy-C₁₋₄alkoxy group; or adjacent two R³ form, taken together, a C₁₋₃alkylenedioxy group; R⁴ is a hydrogen atom or a C₁₋₃ alkyl group; R⁶ isa C₁₋₄ alkoxy-C₁₋₄ alkyl group or a group of the formula:

 wherein R⁵ is a hydrogen atom or R⁴ and R⁵ form, taken together, a 5-or 6-membered heterocycle; and n is 1 or 2;

[10] a compound of the above [1] or a salt thereof, wherein R¹ is ahydroxy group, a methoxy group or a C₁₋₃ alkyl group; R² is a hydrogenatom or a C₁₋₃ alkyl group; R⁴ is a C₁₋₃ alkyl group; R⁶ is a benzylgroup; and n is 0;

[11]5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dioneor a salt thereof;

[12] a process for producing a compound of claim 1 or a salt thereof,which comprises reacting a compound of the formula:

 wherein each symbol is as defined above, or a salt thereof [hereinaftersometimes referred to briefly as compound (II)] with carbonyldiimidazoleor phosgene, followed by reacting with a compound of the formula:

 wherein each symbol is as defined above, or a salt thereof [hereinaftersometimes referred to briefly as compound (III)];

[13] a pharmaceutical composition which comprises a compound of theabove [1] or a salt thereof;

[14] a pharmaceutical composition of the above [13] which is forantagonizing gonadotropin-releasing hormone;

[15] a pharmaceutical composition of the above [14] which is forpreventing or treating a sex hormone dependent disease;

[16] a method for antagonizing gonadotropin-releasing hormone in amammal in need thereof which comprises administering to said mammal aneffective amount of a compound of the above [1] or a salt thereof with apharmaceutically acceptable excipient, carrier or diluent;

[17] use of a compound of the above [1] or a salt thereof formanufacturing a pharmaceutical composition for antagonizinggonadotropin-releasing hormone, and so forth.

Each symbol in the above formulae is hereinafter described in moredetail.

The “C₁₋₄ alkoxy group” for R¹ or R² includes, for example, methoxy,ethoxy, propoxy, butoxy, isopropoxy, t-butoxy, etc. Among others,preferred is C₁₋₃ alkoxy. More preferred is methoxy.

The “C₁₋₄ alkoxy-carbonyl group” for R¹ or R² includes, for example,methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, t-butoxycarbonyl, etc. Among others, preferred is C₁₋₃alkoxy-carbonyl. More preferred is methoxycarbonyl.

The “C₁₋₄ alkyl group” of the “C₁₋₄ alkyl group which may besubstituted” for R¹ or R² includes, for example, a straight-chain C₁₋₄alkyl group (e.g., methyl, ethyl, propyl, butyl, etc.), a branched C₃₋₄alkyl group (e.g., isopropyl, isobutyl, sec-butyl, tert-butyl, etc.),and so forth. Among others, preferred is a C₁₋₃ alkyl group. Morepreferred is ethyl.

The “substituents” of the “C₁₋₄ alkyl group which may be substituted”for R¹ or R² include, for example, (i) hydroxy, (ii) C₁₋₇ acyloxy (e.g.,C₁₋₆ alkyl-carbonyloxy such as acetoxy, propionyloxy, etc.), (iii)benzoyloxy, (iv) amino which may be substituted by 1 or 2 substituentsselected from the group consisting of C₁₋₆ alkoxy-carbonyl (e.g.,methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, etc.),benzyloxycarbonyl, C₁₋₄ acyl (e.g., C₁₋₃ alkyl-carbonyl such as acetyl,propionyl, etc.), C₁₋₄ alkyl (e.g., methyl, ethyl, propyl, butyl, etc.)and C₁₋₃ alkylsulfonyl (e.g., methanesulfonyl etc.), etc. [e.g., amino,dimethylamino, methoxycarbonylamino, ethoxycarbonylamino,tert-butoxycarbonylamino, benzyloxycarbonylamino, acetylamino,methanesulfonylamino, etc.), (v) C₁₋₁₀ alkoxy (e.g., methoxy, ethoxy,propoxy, tert-butoxy, etc.), (vi) C₃₋₇ cycloalkyloxycarbonyl-C₁₋₃ alkoxy(e.g., cyclohexyloxycarbonyloxy-1-ethoxy, etc.), (vii) C₁₋₃ alkoxy-C₁₋₃alkoxy (e.g., methoxymethoxy, methoxyethoxy, etc.), and so forth. Amongothers, preferred is hydroxy.

The “C₁₋₄ alkyl group” of the “C₁₋₄ alkyl group which may besubstituted” for R¹ or R² may have 1 to 5, preferably 1 to 3,substituents as mentioned above at possible positions and, when thenumber of substituents is two or more, those substituents may be thesame as or different from one another.

Preferably, one of R¹ and R² is a hydrogen atom, and the other is a C₁₋₃alkoxy group.

The “halogen atom” for R³ includes, for example, fluoro, chloro, bromo,iodo, etc. Among others, preferred is chloro.

The “C₁₋₄ alkoxy group” of the “C₁₋₄ alkoxy group which may besubstituted” for R³ includes, for example, methoxy, ethoxy, propoxy,isopropoxy, butoxy, t-butoxy, etc. Among others, preferred is methoxy.

The “substituents” of the “C₁₋₄ alkoxy group which may be substituted”for R³ are the same as those mentioned above for the “substituents” ofthe “C₁₋₄ alkyl group which may be substituted” for R¹ or R². Amongothers, preferred is a C₁₋₄ alkoxy group.

The “C₁₋₄ alkoxy group” may have 1 to 5, preferably 1 to 3, substituentsas mentioned above at possible positions and, when the number ofsubstituents is two or more, those substituents may be the same as ordifferent from one another.

The “C₁₋₄ alkylenedioxy group” formed by adjacent two R³ includes, forexample, methylenedioxy, ethylenedioxy, etc.

R³ is preferably a hydrogen atom.

The “C₁₋₄ alkyl group” for R⁴ includes, for example, a straight-chainC₁₋₄ alkyl group (e.g methyl, ethyl, propyl, butyl, etc.), a branchedC₃₋₄ alkyl group (e.g., isopropyl, isobutyl, sec-butyl, tert-butyl,etc.), and so forth. Among others, preferred is a C₁₋₃ alkyl group. Morepreferred is methyl.

The “C₁₋₄ alkyl group which may be substituted” for R⁶ are the same asthose mentioned above for the “C₁₋₄ alkyl group which may besubstituted” for R¹ or R².

The “heterocycle” formed by R⁴ and R⁵ includes, for example, a 5- or6-membered N-containing heterocycle, etc. When R⁴ and R⁵ form, takentogether, examples of the group of the formula:

include a group of the formula:

Among others, preferred is a group of the formula:

Preferably, R⁶ is a group of the formula:

wherein R⁵ is as defined above.

Preferably, R⁴ is C₁₋₃ alkyl and R⁵ is a hydrogen atom.

Preferably, n is 1 or 2.

Preferable examples of compound (I) include a compound (Ia).

More preferred is a compound or a salt thereof, wherein R¹ is a hydroxygroup, a methoxy group or a C₁₋₃ alkyl group; R² is a hydrogen atom or aC₁₋₃ alkyl group; R⁴ is a C₁₋₃ alkyl group; R⁶ is a benzyl group; and nis 0.

Among others, more preferred is a compound or a salt thereof, wherein R¹is a C₁₋₃ alkoxy group; R² and R⁵ each is a hydrogen atom; R⁴ is a C₁₋₃alkyl group; R⁶ is a benzyl group; and n is 0.

Other preferable examples of compound (I) include a compound or a saltthereof, wherein R¹ is (i) a hydroxy group, (ii) a C₁₋₄ alkoxy group, or(iii) a C₁₋₄ alkyl group which may be substituted by hydroxy or C₁₋₄alkyl-carbonyloxy; R² is a hydrogen atom, a C₁₋₄ alkyl group or a C₁₋₄alkoxy-carbonyl group; R³ is a hydrogen atom, a halogen atom, a hydroxygroup or a C₁₋₄ alkoxy-C₁₋₄ alkoxy group; or adjacent two R³ form, takentogether, a C₁₋₃ alkylenedioxy group; R⁴ is a hydrogen atom or a C₁₋₃alkyl group; R⁶ is a C₁₋₄ alkoxy-C₁₋₄ alkyl group or a group of theformula:

wherein R⁵ is a hydrogen atom or R⁴ and R⁵ form, taken together, a 5- or6-membered heterocycle; and n is 1 or 2.

As compound (I), concretely mentioned are

5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dioneor a salt thereof,

5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-hydroxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dioneor a salt thereof,

5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methylureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dioneor a salt thereof,

5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-ethylureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dioneor a salt thereof, and so forth.

Among others, preferred is5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-drpyrimidine-2,4(1H,3H)-dioneor a salt thereof.

Salts of compound (I) are preferably physiologically acceptable acidaddition salts. Such salts include, for example, salts with inorganicacids (e.g., hydrochloric acid, hydrobromic acid, nitric acid, sulfuricacid, phosphoric acid), salts with organic acids (e.g., formic acid,acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaricacid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,etc.), and so forth. When compound (I) has an acidic group, it may forma physiologically acceptable salt with an inorganic base (e.g., alkalimetals and alkaline earth metals such as sodium, potassium, calcium andmagnesium, ammonia) or an organic base (e.g., trimethylamine,triethylamine, pyridine, picoline, ethanolamine, diethanolamine,triethanolamine, dicyclohexylamine, N,N′-dibenzylethylenediamine, etc).

Compound (I) can be produced in any per se known manner, for example,according to the methods disclosed in JP-A-9-169768, WO 96/24597 oranalogous methods thereto. Concretely mentioned are the followingProduction method 1 and Production method 2. Compounds (II) to (VII)described in the following process include their salts. For their salts,for example, referred to are the same as the salts of compound (I).

Production Method 1

In the above formulae, L represents a leaving group, and other symbolsare as defined above.

The “leaving group” for L includes, for example, 1-imidazolyl, halogen,an alkoxy group which may be substituted, etc. The “alkoxy group whichmay be substituted” includes, for example, a C₁₋₄ alkoxy group which maybe substituted by 1 to 3 halogen such as chloro, bromo, etc. (e.g.,2,2,2-trichloroethoxy, etc.).

Compound (II) can be produced by the methods disclosed in JP-A-9-169768or analogous methods thereto.

Compound (I) can be produced by reacting compound (II) withcarbonyldiimidazole (N,N′-carbonyldiimidazole; CDI) or phosgene(monomer, dimer or trimer) to obtain compound (IV), followed by reactingwith compound (III). The reaction can be carried out without isolationof compound (IV), or compound (IV) can be used as a purified form in thenext reaction.

Compound (IV) can also be produced by reacting compound (II) with, forexample, a chloroformic acid ester compound (e.g., chloroformic acid2,2,2-trichloroethyl ester, chloroformic acid 1-chloroethyl ester,etc.).

In the reaction of compound (II) with carbonyldiimidazole or phosgene,etc., carbonyldiimidazole or phosgene, etc. is used in amount of about 1to 3 moles, relative to one mole of compound (II).

This reaction is advantageously carried out in a solvent inert to thereaction.

Examples of the solvent include ethers (e.g., ethyl ether, dioxane,dimethoxyethane, tetrahydrofuran, etc.), aromatic hydrocarbons (e.g.,benzene, toluene, etc.) amides (e.g., dimethylformamide,dimethylacetamide, etc.), halogenated hydrocarbons (e.g., chloroform,dichloromethane, etc.), and so forth.

The reaction temperature is usually about 0 to 150° C., preferably roomtemperature (about 15 to 25° C.). The reaction time is usually about 1to 36 hours.

This reaction is also carried out in the presence of a base. The “base”is exemplified by inorganic bases such as sodium carbonate, sodiumhydrogencarbonate, potassium carbonate, potassium hydrogencarbonate,sodium hydroxide, potassium hydroxide and thallium hydroxide, andorganic bases such as triethylamine and pyridine, etc.

The amount of the “base” is about 2 to 20 moles, preferably about 5 to12 moles, relative to one mole of compound (II).

The following reaction with compound (III) can be carried out in thesame condition as the above reaction of compound (II) withcarbonyldiimidazole or phosgene. The amount of compound (III) is about 2to 20 moles, preferably about 5 to 10 moles, relative to one mole ofcompound (II) or compound (IV). The reaction temperature is usuallyabout 0 to 150° C., preferably room temperature (about 15 to 25° C.).The reaction time is usually about 1 to 6 hours.

Compound (III) and carbonyldiimidazole or phosgene can be reacted withcompound (II) at the same time.

Production Method 2

In the above formulae, R⁷ represents a hydrogen atom or an alkyl group,R⁸ represents an alkyl group, and other symbols are as defined above.

The “C₁₋₄ alkyl group” for R⁷ or R⁸ includes, for example, the “C₁₋₄alkyl group” of the “C₁₋₄ alkyl group which may be substituted” for R¹or R².

Compound (V) can be produced in any per se known manner, for example,p-nitrophenylacetone is reacted with a cyanoacetic ester compound andsulphur [e.g., Chem. Ber., 99, 94-100(1966)], and thus obtained2-amino-4-methyl-5-(4-nitrophenyl)thiophene is subjected to the methodsdisclosed in JP-A-9-169768, WO 96/24597 or analogous methods thereto.

1) When R⁷ is a hydrogen atom, compound (I) can be produced by reactingcompound (V) with a compound of the formula:

wherein each symbol is as defined above, or a salt thereof [hereinaftersometimes referred to briefly as compound (VI)], in the presence of acondensing agent, to obtain compound (VII), following by subjecting tocyclization.

The “condensing agent” includes, for example,benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), etc.

The amount of the “condensing agent” is about 1 to 3 moles, relative toone mole of compound (V).

This reaction is advantageously carried out in a solvent inert to thereaction.

Examples of the solvent include alcohols (e.g., ethanol, methanol,etc.), aromatic hydrocarbons (e.g., benzene, toluene, etc.), amides(e.g., dimethylformamide, dimethylacetamide, etc.), halogenatedhydrocarbons (e.g., chloroform, dichloromethane, etc.), and so forth.

The reaction temperature is usually about 0 to 150° C., preferably roomtemperature (about 15 to 25° C.). The reaction time is usually about 1to 36 hours.

The product as produced in the manner mentioned above may be applied tothe next reaction while it is still crude in the reaction mixture, ormay be isolated from the reaction mixture in any ordinary manner.

Compound (VII) is subjected to cyclization in the presence of a base.

The “base” is exemplified by inorganic bases such as sodium methoxide,sodium carbonate, sodium hydrogencarbonate, potassium carbonate,potassium hydrogencarbonate, sodium hydroxide, potassium hydroxide andthallium hydroxide, and organic bases such as triethylamine andpyridine, etc.

The amount of the “base” is about 2 to 20 moles, preferably about 5 to12 moles, relative to one mole of compound (VII).

This reaction is advantageously carried out in a solvent inert to thereaction.

Examples of the solvent include alcohols (e.g., ethanol, methanol,etc.), aromatic hydrocarbons (e.g., benzene, toluene, etc.), amides(e.g., dimethylformamide, dimethylacetamide, etc.), halogenatedhydrocarbons (e.g., chloroform, dichloromethane, etc.), and so forth.

The reaction temperature is usually about 0 to 150° C., preferably roomtemperature (about 15 to 25° C.). The reaction time is usually about 1to 36 hours.

2) When R⁷ is an alkyl group, compound (I) can be produced by reactingcompound (V) with an activated compound (VI).

The activated compound (VI) can be produced in any per se known manner,for example, by reacting an organo-aluminum reagent with compound (VI)in a solvent inert to the reaction.

The “organo-aluminum reagent” includes, for example, trimethyl aluminum,dimethyl aluminum chloride, etc, and a solution including them, etc.

The amount of the “organo-aluminum reagent” is about 1 to 5 moles,preferably about one mole, relative to one mole of compound (VI).

Examples of the solvent include halogenated hydrocarbons (e.g.,chloroform, dichloromethane, etc.), and so forth.

The reaction temperature is usually about 0 to 150° C., preferably roomtemperature (about 15 to 25° C.). The reaction time is usually about 1to 6 hours.

The cyclization can be carried out by reacting compound (V) with anactivated compound (VI) to obtain compound (I).

The amount of the “compound (V)” is about 1/5 volume of a mixture ofcompound (VI) and the organo-aluminum reagent.

This reaction is advantageously carried out in a solvent inert to thereaction.

Such solvent is the same as those used in the reaction to obtain anactivated compound (VI).

The reaction temperature is usually about 0 to 150° C., preferably roomtemperature (about 15 to 25° C.). The reaction time is usually about 1to 48 hours.

Compound (I) may be isolated and purified by ordinary means ofseparation such as recrystallization, distillation and chromatography,etc.

When compound (I) is obtained in free form, it can be converted to asalt by per se known methods or analogous thereto. When compound (I) isobtained in salt form, it can be converted to the free form or anothersalt by per se known methods or analogous thereto.

Compound (I) may be a hydrate or a non-hydrate. The hydrate isexemplified by monohydrate, sesquihydrate and dehydrate.

When compound (I) is obtained as a mixture of optically activeconfigurations. it can be resolved into the (R)- and (S)-forms by theconventional optical resolution techniques.

Compound (I) maybe labeled by an isotope (e.g., ³H, ¹⁴C, ³⁵S, etc.).

Compound (I) of the present invention (hereinafter also referred to as“compound of the present invention”) possesses excellentGnRH-antagonizing activity and low toxicity. In addition, it isexcellent in oral absorbability, action sustainability, stability andpharmacokinetics. Furthermore, it can be easily produced. The compoundof the present invention can therefore be safely used in a mammal (e.g.,human, monkey, bovine, horse, dog, cat, rabbit, rat, mouse, etc.) forthe preventing and/or treating diseases depending on male or femalehormones, diseases due to excess of these hormones, etc., by suppressinggonadotropin secretion by its GnRH receptor-antagonizing action tocontrol plasma sex hormone concentrations.

For example, the compound of the present invention is useful forpreventing and/or treating sex hormone-dependent cancers (e.g.,prostatic cancer, uterine cancer, breast cancer, pituitary tumor, etc.),prostatic hypertrophy, hysteromyoma, endometriosis, precocious puberty,amenorrhea, premenstrual syndrome, multilocular ovary syndrome, pimplesetc. The compound of the present invention is also useful for theregulation of reproduction in males and females (e. g., pregnancyregulators, menstruation cycle regulators, etc.). The compound of thepresent invention also be used as a male or female contraceptive, or asa female ovulation inducer. Based on its rebound effect afterwithdrawal, the compound of the present invention can be used to treatinfertility.

In addition, the compound of the present invention is useful forregulation of animal estrous, improvement of meat quality and promotionof animal growth in the field of animal husbandry. The compound of thepresent invention is also useful as a fish spawning promoter.

The compound of the present invention can also be used to suppress thetransient rise in plasma testosterone concentration (flare phenomenon)observed in administration of a GnRH super-agonist such as leuprorelinacetate. The compound of the present invention can be used incombination with a GnRH super-agonist such as leuprorelin acetate,gonadrelin, buserelin, triptorelin, goserelin, nafarelin, histrelin,deslorelin, meterelin, lecirelin, and so forth. Among others, preferredis leuprorelin acetate.

It is also beneficial to use the compound of the present invention inconjunction (in combination or concomitantly) with at least one memberselected from among the steroidal or nonsteroidal androgen antagonist orantiestrogen, chemotherapeutic agent, GnRH antagonistic peptide,α-reductase inhibitor, α-receptor inhibitor, aromatase inhibitor,17β-hydroxysteroid dehydrogenase inhibitor, adrenal androgen productioninhibitor, protein kinase inhibitor, drug for hormone therapy, and drugantagonizing growth factor or its receptor, among others.

The “chemotherapeutic agent” mentioned above includes ifosfamide, UTF,adriamycin, peplomycin, cisplatin, cyclophosphamide, 5-FU, UFT,methotrexate, mitomycin C, mitoxantrone, etc.

The “GnRH antagonistic peptide” mentioned above includes non-oral GnRHantagonistic peptides such as cetrorelix, ganirelix, abarelix, etc.

The “adrenal androgen production inhibitor” mentioned above includeslyase (C_(17,20)-lyase) inhibitors, etc.

The “protein kinase inhibitor” mentioned above includes tyrosine kinaseinhibitor, etc.

The “drugs for hormone therapy” includes antiestrogens, progesterons(e.g., MPA, etc.), androgens, estrogens and androgen antagonists, amongothers.

The “growth factor” may be any substance that promotes proliferation ofcells and generally includes peptides with molecular weights not over20,000 which express the action at low concentrations through binding toreceptors. Specifically, there can be mentioned (1) EGF (epidermalgrowth factor) or substances having the substantially the same activity(e.g., EGF, heregulin (HER2 ligand), etc.), (2) insulin or substanceshaving substantially the same activity (e.g., insulin, IGF (insulin-likegrowth factor)-1, IGF-2, etc.), (3) FGF (fibroblast growth factor) orsubstances having substantially the same activity (aFGF, bFGF, KGF(keratinocyte growth factor), HGF (hepatocyte growth factor), FGF-10,etc.), and (4) other growth factors (e.g., CSF (colony stimulatingfactor), EPO (erythropoietin), IL-2 (interleukin-2), NGF (nerve growthfactor), PDGF (platelet-derived growth factor) and TGFβ (transforminggrowth factor β), etc.), among others.

The “growth factor receptor” mentioned above may be any receptor capableof binding said growth factor, including EGF receptor, heregulinreceptor (HER2), insulin receptor-1, insulin receptor-2, IGF receptor,FGF receptor-1, FGF receptor-2, etc.

The drug antagonizing said growth factor includes herceptin (anti-HER2receptor antibody), among others.

The drug antagonizing said growth factor or growth factor receptorincludes herbimycin, PD153035 [e.g., Science, 265 (5175) p1093, (1994)],etc. can be mentioned.

As a further class of drugs antagonizing said growth factor or growthfactor receptor includes HER2 antagonists. The HER2 antagonist may beany substance that inhibits the activity of HER2 (e.g., phosphorylatingactivity), thus including an antibody, a low-molecular compound(synthetic or natural product), an antisense, an HER2 ligand, heregulin,and any of them as partially modified or mutated in structure. Moreover,it may be a substance which inhibits HER2 activity by antagonizing HER2receptor (e.g. HER2 receptor antibody). The low molecular compoundhaving HER2 antagonizing activity includes, for example, the compoundsdescribed in WO 98/03505, namely1-[3-[4-[2-((E)-2-phenylethenyl)-4-oxazolylmethoxy]phenyl]propyl]-1,2,4-triazoleand so on.

For prostatic hypertrophy, examples of such combination includes thecompound of the present invention in combination with the GnRHsuper-agonist, androgen antagonist, antiestrogen, GnRH antagonisticpeptide, α-reductase inhibitor, α-receptor inhibitor, aromataseinhibitor, 17β-hydroxysteroid dehydrogenase inhibitor, adrenal androgenproduction inhibitor, phosphorylase inhibitor, and so forth.

For prostatic cancer, examples of such combination includes the compoundof the present invention in combination with the GnRH super-agonist,androgen antagonist, antiestrogen, chemotherapeutic agent (e.g.,ifosfamide, UTF, adriamycin, peplomycin, cisplatin, etc.), GnRHantagonistic peptide, aromatase inhibitor, 17β-hydroxysteroiddehydrogenase inhibitor, adrenal androgen production inhibitor,phosphorylase inhibitor, drug for hormone therapy such as estrogens(e.g., DSB, EMP, etc.), androgen antagonist (e.g., CMA. etc.), drugantagonizing growth factor or its receptor, and so forth.

For breast cancer, examples of such combination includes the compound ofthe present invention in combination with the GnRH super-agonist,chemotherapeutic agent (e.g., cyclophosphamide, 5-FU, UFT, methotrexate,adriamycin, mitomycin C, mitoxantrone, etc.), GnRH antagonistic peptide,aromatase inhibitor, adrenal androgen production inhibitor,phosphorylase inhibitor, drug for hormone therapy such as antiestrogen(e.g., tamoxifen, etc.), progesterons (e.g., MPA, etc.), androgens,estrogens, etc., drug antagonizing growth factor or its receptor, and soforth.

When the compound of the present invention is used as a prophylacticand/or therapeutic agent for the above-mentioned diseases or used in thefield of animal husbandry or fishery, it can be administered orally ornon-orally, as formulated with a pharmaceutically acceptable carrier,normally in the form of solid preparations such as tablets, capsules,granules and powders for oral administration, or in the form ofintravenous, subcutaneous, intramuscular or other injections,suppositories or sublingual tablets for non-oral administration. It mayalso be sublingually, subcutaneously, intramuscularly or otherwiseadministered in the form of sustained-release preparations of sublingualtablets, microcapsules etc. Depending on symptom severity; subject age,sex, weight and sensitivity; duration and intervals of administration;property, dispensing and kind of pharmaceutical preparation; kind ofactive ingredient etc., daily dose is not subject to limitation. For usein the treatment of the above-described sex hormone-dependent cancers(e.g., prostatic cancer, uterine cancer, breast cancer, pituitarytumor), prostatic hypertrophy, hysteromyoma, endometriosis, precociouspuberty etc., daily dose is normally about 0.01 to 30 mg, preferablyabout 0.02 to 10 mg, and more preferably 0.1 to 10 mg, especiallypreferably 0.1 to 5 mg per kg weight of mammal, normally in 1 to 4divided dosages.

The above doses are applicable to the use of the compound of the presentinvention in the field of animal husbandry or fishery. Daily dose isabout 0.01 to 30 mg, preferably about 0.1 to 10 mg, per kg weight ofsubject organism, normally in 1 to 3 divided dosages.

In the pharmaceutical composition of the present invention, the amountof compound (I) is 0.01 to 100% by weight or so of the total weight ofthe composition.

The above pharmaceutically acceptable carriers are various organic orinorganic carrier substances in common use as pharmaceutical materials,including excipients, lubricants, binders and disintegrants for solidpreparations, and solvents, dissolution aids, suspending agents,isotonizing agents, buffers and soothing agents for liquid preparations.Other pharmaceutical additives such as preservatives, antioxidants,coloring agents and sweetening agents may be used as necessary.

Preferable excipients include, for example, lactose, sucrose,D-mannitol, starch, crystalline cellulose and light silicic anhydride.Preferable lubricants include, for example, magnesium stearate, calciumstearate, talc and colloidal silica. Preferable binders include, forexample, crystalline cellulose, sucrose, D-mannitol, dextrin,hydroxypropyl cellulose, hydroxypropylmethyl cellulose andpolyvinylpyrrolidone. Preferable disintegrants include, for example,starch, carboxymethyl cellulose, carboxymethyl cellulose calcium,crosslinked carmellose sodium and carboxymethyl starch sodium.Preferable solvents include, for example, water for injection, alcohol,propylene glycol, macrogol, sesame oil and corn oil. Preferabledissolution aids include, for example, polyethylene glycol, propyleneglycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane,cholesterol, triethanolamine, sodium carbonate and sodium citrate.Preferable suspending agents include, for example, surfactants such asstearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionicacid, lecithin, benzalkonium chloride, benzethonium chloride andmonostearic glycerol; and hydrophilic polymers such as polyvinylalcohol, polyvinylpyrrolidone, carboxymethyl cellulose sodium, methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose andhydroxypropyl cellulose. Preferable isotonizing agents include, forexample, sodium chloride, glycerol and D-mannitol. Preferable buffersinclude, for example, buffer solutions of phosphates, acetates,carbonates, citrates etc. Preferable soothing agents include, forexample, benzyl alcohol. Preferable preservatives include, for example,paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethylalcohol, dehydroaceticacidandsorbic acid. Preferable antioxidantsinclude, for example, sulfites and ascorbic acid.

By adding suspending agents, dissolution aids, stabilizers, isotonizingagents, preservatives, and so forth, the compound of the presentinvention can be prepared as an intravenous, subcutaneous orintramuscular injection by a commonly known method. In such cases, thecompound of the present invention can be freeze-dried as necessary by acommonly known method. In administration to humans, for example, thecompound of the present invention can be safely administered orally ornon-orally as such or as a pharmaceutical composition prepared by mixingit with a pharmacologically acceptable carrier, excipient and diluentselected as appropriate.

Such pharmaceutical compositions include oral preparations (e.g.,powders, granules, capsules, tablets), injections, drip infusions,external preparations (e.g., nasal preparations, transdermalpreparations) and suppositories (e.g., rectal suppositories, vaginalsuppositories).

These preparations can be produced by commonly known methods in commonuse for pharmaceutical making processes.

An injection can be produced by, for example, preparing the compound ofthe present invention as an aqueous injection along with a dispersingagent (e.g., Tween 80, produced by Atlas Powder Company, USA, HCO 60,produced by Nikko Chemicals Co., Ltd., polyethylene glycol,carboxymethyl cellulose, sodium alginate), a preservative (e.g., methylparaben, propyl paraben, benzyl alcohol), an isotonizing agent (e.g.,sodium chloride, mannitol, sorbitol, glucose) and other additives, or asan oily injection in solution, suspension or emulsion in a vegetable oilsuch as olive oil, sesame oil, cottonseed oil or corn oil, propyleneglycol or the like.

An oral preparation can be produced by formulating the compound of thepresent invention by a commonly known method after addition of anexcipient (e.g., lactose, sucrose, starch), a disintegrant (e.g.,starch, calcium carbonate), a binder (e.g., starch, gum arabic,carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose),a lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000)and other additives, and, where necessary, coating the formulatedproduct for the purpose of taste masking, enteric dissolution orsustained release by a commonly known method. Coating agents for thispurpose include, for example, hydroxypropylmethyl cellulose, ethylcellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,polyoxyethylene glycol, Tween 80, Prulonic F68, cellulose acetatephthalate, hydroxypropylmethyl cellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (produced by Rohm Company,Germany; methacrylic acid/acrylic acid copolymer) and dyes (e.g., ironoxide, titanium dioxide). For an enteric preparation, an intermediatephase may be provided between the enteric phase and the drug-containingphase for the purpose of separation of the two phases by a commonlyknown method.

An external preparation can be produced by compounding the compound ofthe present invention as a solid, semi-solid or liquid composition by acommonly known method. Such a solid composition is produced by, forexample, powdering the compound of the present invention as such or inmixture with an excipient (e.g., glycol, mannitol, starch,microcrystalline cellulose), a thickening agent (e.g., natural rubber,cellulose derivative, acrylic acid polymer) and other additives. Such aliquid composition is produced by preparing the compound of the presentinvention as an oily or aqueous suspension in almost the same manner aswith the injection. The semi-solid composition is preferably an aqueousor oily gel, or an ointment. All these compositions may contain pHregulators (e.g., carbonic acid, phosphoric acid, citric acid,hydrochloric acid, sodium hydroxide), preservatives (e.g.,paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride) andother additives.

A suppository is produced by preparing the compound of the presentinvention as an oily or aqueous solid, semi-solid or liquid compositionby a commonly known method. Useful oily bases for such compositionsinclude glycerides of higher fatty acids (e. g., cacao fat, uitepsols,produced by Dynamite Nobel Company, Germany), moderate fatty acids(e.g., MIGLYOL, produced by Dynamite Nobel Company, Germany), andvegetable oils (e.g., sesame oil, soybean oil, cottonseed oil). Aqueousbases include, for example, polyethylene glycols and propylene glycol.Bases for aqueous gels include, for example, natural rubbers, cellulosederivatives, vinyl polymers and acrylic acid polymers.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is hereinafter described in more detail by meansof, but is not limited to, the following reference examples, examples,preparation examples and experimental examples.

¹H-NMR spectra are determined with tetramethylsilane as the internalstandard, using the Varian GEMINI 200 (200 MHz) spectrometer, the JEOLLAMBDA 300 (300 MHz) spectrometer or the Bruker AM500 (500 MHz)spectrometer; all δ values are shown in ppm. Unless otherwisespecifically indicated, “%” is by weight. Yield indicates mol/mol %.

The other symbols used herein have the following definitions:

s: singlet

d: doublet

t: triplet

dt: double triplet

m: multiplet

br: broad

TFA: trifluoroacetic acid

THF: tetrahydrofuran

Me: methyl

Et: ethyl

The term “room temperature” indicates the range from about 15 to 25° C.,but is not to be construed as strictly limitative.

EXAMPLES Reference Example 1

Ethyl 2-amino-4-methyl-5-(4-nitrophenyl)thiophene-3-carboxylate

A mixture of 4-nitrophenylacetone (35.0 g, 195 mmol), ethyl cyanoacetate(23.8 g, 195 mmol), ammonium acetate (3.1 g, 40 mmol) and acetic acid(9.1 ml, 159 mmol) was heated on reflux for 24 hours, with removingwater produced through the reaction with a Dean-Stark trap. Aftercooling, the reaction mixture was concentrated under reduced pressureand the residue was partitioned between dichloromethane and aqueoussodium hydrogencarbonate solution. The organic extract was washed withaqueous sodium chloride solution and dried (MgSO₄) and the solvent wasdistilled off under reduced pressure. The residue was chromatographed onsilica gel to give oil compound. The oil thus obtained was dissolved inethanol followed by addition of sulfur (5.0 g, 160 mmol) anddiethylamine (16.0 ml, 160 mmol), and the mixture was stirred at 60 to70° C. for 2 hours. After cooling, the reaction mixture was concentratedunder reduced pressure to yield residue, which was partitioned betweendichloromethane and aqueous sodium hydrogencarbonate solution. Theorganic extract was washed with aqueous sodium chloride solution anddried (MgSO₄) and the solvent was distilled off under reduced pressure.The residue was chromatographed on silica gel to give the crude product,which was recrystallized from ether-hexane to give the title compound asred plates (22.2 g, 52%).

mp: 168-170° C. (recrystallized from ether-hexane). Elemental analysisfor C₁₄H₁₄N₂O₄S; C (%) H (%) N (%); Calculated: 54.89; 4.61; 9.14;Found: 54.83; 4.90; 9.09; ¹H-NMR (200 MHz, CDCl₃) δ: 1.39 (3H, t, J=7.1Hz), 2.40 (3H, s), 4.34 (2H, q, J=7.1 Hz), 6.27 (2H, br), 7.48 (2H, d,J=8.7Hz), 8.23 (2H, d, J=8.7Hz). IR (KBr): 3446, 3324, 1667, 1580, 1545,1506, 1491, 1475, 1410, 1332 cm⁻¹.

Reference Example 2

5-Methyl-6-(4-nitrophenyl)-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of the compound obtained in Reference Example 1 (5.00 g,16.32 mmol) in pyridine (30 ml) was added phenyl isocyanate (2.66 ml,24.48 mmol). After 6 hours of stirring at 45° C., the reaction mixturewas concentrated under reduced pressure and the residue was dissolved inethanol (6 ml). To this solution was added 28% sodium methoxide (7.86 g,40.80 mmol), and the mixture was stirred at room temperature for 2hours. Then, 2N-hydrochloric acid (25 ml, 50 mmol) was added and thesolvent ethanol was distilled off under reduced pressure. The residuewas filtered, washed with water-ethanol, dried in vacuo, andrecrystallized from ethanol to give the title compound as yellow powder(6.09 g, 98%).

mp: >300° C.; Elemental analysis for C₁₉H₁₃N₃O₄S.0.3H₂O; C(%) H(%) N(%);Calculated: 59.30; 3.56; 10.92; Found: 59.56; 3.52; 10.93; ¹H-NMR (300MHz, DMSO-d₆) δ: 2.50 (3H, s), 7.31-7.46 (5H, m), 7.78 (2H, d, J=8.8Hz), 8.32 (2H, d, J=8.8 Hz), 12.50 (1H, s). IR (KBr): 1715, 1657, 1593,1510 cm⁻¹.

Reference Example 3

1-(2,6-Difluorobenzyl)-5-methyl-6-(4-nitrophenyl)-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of the compound obtained in Reference Example 2 (52.54 g,0.131 mmol) in N,N-dimethylformamide (1.0 L) were added potassiumcarbonate (19.00 g, 0.138 mol), potassium iodide (22.90 g, 0.138 mol)and 2,6-difluorobenzyl chloride (22.40 g, 0.138 mol), and the mixturewas stirred at room temperature for 2 hours. This reaction mixture wasconcentrated under reduced pressure to give the residue, which waspartitioned between chloroform and aqueous sodium chloride solution. Theaqueous layer was extracted with chloroform. The combined extracts werewashed with aqueous sodium chloride solution and dried (MgSO₄) and thesolvent was distilled off under reduced pressure. The residue waschromatographed on silica gel to give the title compound as light-yellowcrystals (61.50 g, 93%).

mp: 280-282° C.; Elemental analysis for C₂₆H₁₇N₃O₄SF₂; C(%) H(%) N(%);Calculated: 61.78; 3.39; 8.31; Found: 61.67; 3.46; 8.21; ¹H-NMR (300MHz, CDCl₃) δ: 2.57 (3H, s), 5.38 (2H, s), 6.94 (2H, d, J=8.1 Hz),7.42-7.58 (8H, m), 8.29 (2H, d, J=8.8 Hz). IR (KBr): 1719, 1669, 1524.,1473 cm⁻¹.

Reference Example 4

5-Bromomethyl-1-(2,6-difluorobenzyl)-6-(4-nitrophenyl)-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

A mixture of the compound obtained in Reference Example 3 (30.34 g,0.060 mol), N-bromosuccinimide (12.81 g, 0.072 mol),α,α′-azobisisobutyronitrile (1.15 g, 0.007 mol) and chlorobenzene (450ml) was stirred at 85° C. for 3 hours. After cooling, the reactionmixture was washed with aqueous sodium chloride solution and dried(MgSO₄) and the solvent was then distilled off under reduced pressure.The residue was recrystallized from ethyl acetate to give the titlecompound as yellow needles (80.21 g, 100%).

mp: 228-229° C.; ¹H-NMR (300 MHz, CDCl₃) δ: 4.77 (2H, s), 5.38 (2H, s),6.96 (2H, t, J=8.1 Hz), 7.29-7.58 (6H, m), 7.79 (2H, d, J=8.5 Hz), 8.35(2H, d, J=8.5 Hz). IR (KBr): 1721, 1680, 1524, 1473, 1348 cm⁻¹. FAB-Massm/z 584(MH)⁺.

Reference Example 5

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-(4-nitrophenyl)-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of the compound obtained in Reference Example 4 (80.00 g,0.119 mol) in N,N-dimethylformamide (600 ml) were addedethyldiisopropylamine (27.00 ml, 0.155 mol) and benzylmethylamine (18.45ml, 0.143 mol) with ice-cooling. After 2 hours of stirring at roomtemperature, the reaction mixture was concentrated and the residue waspartitioned between ethyl acetate and saturated aqueous sodiumhydrogencarbonate solution. The aqueous layer was extracted with ethylacetate. The organic extracts were combined and dried (MgSO₄) and thesolvent was distilled off under reduced pressure. The residue waschromatographed on silica gel to give a yellow oil (74.90 g, 100%),which was recrystallized from ethyl acetate to give the title compoundas yellow needles.

mp: 173-174° C.; Elemental analysis for C₃₄H₂₆N₄O₄SF₂.0.5H₂O; C(%) H(%)N(%); Calculated: 64.45; 4.29; 8.84; Found: 64.50; 4.24; 8.82; ¹H-NMR(300 MHz, CDCl₃) [Free amine] δ: 1.31 (3H, s), 3.60 (2H, s), 3.96 (2H,s), 5.39 (2H, s), 6.95 (2H, t, J=8.2 Hz), 7.18-7.55 (11H, m), 8.02 (2H,d, J=9.0 Hz), 8.26 (2H, d, J=9.0 Hz). IR (KBr) [Hydrochloride]: 1719,1678, 1597, 1520 cm⁻¹.

Reference Example 6

6-(4-Aminophenyl)-5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of the compound obtained in Reference Example 5 (3.00 g,4.80 mmol) in formic acid (30 ml) were added 1M hydrogen chloridesolution in ether (14.4 ml, 14.4 mmol) and 10% palladium-on-carbon (300mg) with ice-cooling, and hydrogenation was carried out underatmospheric condition at room temperature with constant stirring for 2hours. This reaction mixture was filtered through Celite and thefiltrate was concentrated under reduced pressure. The residue waspartitioned between dichloromethane and saturated aqueous sodiumhydrogencarbonate solution. The aqueous layer was extracted withdichloromethane and the organic extracts were combined and dried(MgSO₄). The solvent was then distilled off under reduced pressure. Theresidue was chromatographed on silica gel to give the title compound aswhite crystals (2.41 g, 84%).

mp: 205-207° C.; Elemental analysis for C₃₄H₂₈N₄O₂SF₂.0.1AcOEt.1.2H₂O;C(%) H(%) N(%); Calculated 66.09; 5.03; 8.96; Found 66.93; 4.94; 8.67;¹H-NMR (300 MHz, CDCl₃) δ: 2.05(3H, s), 3.56(2H, s), 3.83(2H, br),3.88(2H, s), 5.36(2H, s), 6.70(2H, d, J=8.8 Hz), 6.88-6.94(2H, m),7.21-7.31(8H, m), 7.41-7.53(5H, m). IR (KBr): 1715, 1657, 1628, 1537cm⁻¹.

Reference Example 7

5-Chloromethyl-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of the Example Compound No. 1 described below (2.00 g,3.00 mmol) in tetrahydrofuran (90 ml) was added 1-chloroethylchloroformate (0.42 ml, 3.89 minol) at −78° C. The reaction mixture wasallowed to warm to room temperature, the mixture was stirred for 2hours. This reaction mixture was partitioned between chloroform andaqueous sodium chloride solution and the aqueous layer was extractedwith chloroform. The extracts were combined, washed with aqueous sodiumchloride solution and dried (MgSO₄) and the solvent was distilled offunder reduced pressure. The residue was chromatographed on silica gel togive the title compound as white powder (1.68 g, 96%).

mp: 217-219° C.; ¹H-NMR (300 MHz, CDCl₃) δ: 3.83 (3H, s), 4.84 (2H, s),5.37 (2H, s), 6.94 (2H, t, J=8.2 Hz), 7.15 (1H, S), 7.28-7.65 (11H, m).IR (KBr): 1717, 1671, 1628, 1541, 1508, 1473 cm⁻¹. FAB-Mass m/z583(MH)⁺.

Reference Example 8

Using the compound obtained in Reference Example 6 as a startingmaterial, the following Reference Example Compounds No. 8-1 to 8-3 wereobtained in the same manner as Examples 1 and 2 described below.

Reference Example Compound No. 8-1:

Yield: 64% mp: 190-194° C.;

Reference Example Compound No. 8-2:

Yield: 91% mp: 210-215° C.

Reference Example Compound No. 8-3:

Yield: 82% mp: 254-257° C.

Reference Example 9

Ethyl2-ethoxycarbonylamino-4-methyl-5-(4-nitrophenyl)thiophene-3-carboxylate

The compound obtained in Reference Example 1 (500 mg, 1.63 mmol) wasdissolved in toluene (9 ml) followed by addition of ethyl chloroformate(0.19 ml, 1.96 mmol), and the mixture was heated under reflux for 5hours. After cooling, the reaction mixture was concentrated underreduced pressure. The residue was chromatographed on silica gel to givethe title compound as yellow powder (90 mg, 79%).

mp: 130-131° C. (recrystallized from ethyl acetate-hexane). ¹H-NMR (200MHz, CDCl₃) δ: 1.35 (3H, t, J=7.1 Hz), 1.42 (3H, t, J=7.2 Hz), 2.42 (3H,s), 4.31 (2H, q, J=7.1 Hz), 4.39 (2H, q, J=7.2 Hz), 7.59 (2H, d, J=9.0Hz), 8.27 (2H, d, J=9.0 Hz), 10.66 (1H, s). IR (KBr): 1740, 1665, 1597,1557, 1533, 1516, 1352,1257 cm⁻¹.

Reference Example 10

Ethyl2-[N-(2,6-difluorobenzyl)-N-ethoxycarbonylamino]-4-methyl-5-(4-nitrophenyl)thiophene-3-carboxylate

To a solution of the compound obtained in Reference Example 9 (490 mg,1.30 mol) in N,N-dimethylformamide (20 ml) were added potassiumcarbonate (196 mg, 1.42 mol), potassium iodide (236 mg, 1.42 mol) and2,6-difluorobenzyl chloride (232 mg, 1.42 mmol), and the mixture wasstirred at room temperature for 5 hours. This reaction mixture wasconcentrated and the residue was partitioned between chloroform andaqueous sodium chloride solution. The aqueous layer was extracted withchloroform. The organic extracts were combined and washed with aqueoussodium chloride solution and dried (MgSO₄) and the solvent was distilledoff under reduced pressure. The residue was chromatographed on silicagel and the amorphous powder obtained was recrystallized from methanolto give the title compound as yellow powdery crystals (520 mg, 79%).

mp: 91-92° C.; ¹H-NMR (300 MHz, CDCl₃) δ: 1.15-1.35 (6H, m), 2.40 (3H,s), 4.15-4.29 (4H, m), 4.97 (2H, s), 6.86 (2H, t, J=7.8 Hz), 7.25-7.32(1H, m), 7.51 (2H, d, J=8.8 Hz), 8.25 (2H, d). IR (KBr): 1717, 1597,1524, 1475, 1392, 1348 cm⁻¹.

Reference Example 11

Ethyl4-bromomethyl-2-[N-(2,6-difluorobenzyl)-N-ethoxycarbonylamino]-5-(4-nitrophenyl)thiophene-3-carboxylate

A mixture of the compound obtained in Reference Example 10 (20 g, 39.64mol), N-bromosuccinimide (7.76 g, 43.60 mol),α,α′-azobisisobutyronitrile (0.72 g, 4.36 mol) and carbon tetrachloride(300 ml) was stirred at 100° C. for 2 hours. After cooling, thisreaction mixture was washed with aqueous sodium chloride solution anddried (MgSO₄) and the solvent was distilled off under reduced pressure.The residue was chromatographed on silica gel to give the title compoundas amorphous powder (23 g, 100%).

mp: 105-108° C.; ¹H-NMR (300 MHz, CDCl₃) δ: 1.15-1.39 (6H, m), 4.09-4.39(4H, m), 4.71 (2H, s), 4.99 (2H, s), 6.86 (2H, t, J=7.8 Hz), 7.22-7.32(1H, m), 7.72 (2H, d, J=8.0 Hz), 8.32 (2H, d, J=8.0 Hz). IR (KBr): 1725,1628, 1522, 1475, 1379, 1348 cm⁻¹. FAB-Mass m/z 582 (MH⁺).

Reference Example 12

Ethyl4-(N-benzyl-N-methylaminomethyl)-2-[N-(2,6-difluorobenzyl)-N-ethoxycarbonylamino]-5-(4-nitrophenyl)thiophene-3-carboxylate

To a solution of the compound obtained in Reference Example 11 (2.0 g,3.43 mmol) in N,N-dimethylformamide (20 ml) were addedethyldiisopropylamine (0.90 ml, 5.15 mmol) and benzylmethylamine (0.53ml, 4.11 mmol) with ice-cooling, and the mixture was stirred at roomtemperature for 3 hours. This reaction mixture was concentrated and theresidue was partitioned between ethyl acetate and saturated aqueoussodium hydrogencarbonate solution. The aqueous layer was extracted withethyl acetate. The organic extracts were combined and dried (MgSO₄) andthe solvent was distilled off under reduced pressure. The residue waschromatographed on silica gel to give the title compound as yellow oil(2.1 g, 48%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.18-1.44 (6H, m), 1.95 (3H, s), 3.27 (2H,s), 3.70 (2H, s), 4.20-4.32 (4H, m), 5.03 (2H, s), 6.80 (2H, t, J=7.8Hz), 7.10-7.27 (6H, m), 7.52 (2H, d, J=8.0 Hz), 8.24 (2H, d, J=8.0 Hz).IR (KBr)1719, 1628, 1597, 1522, 1473, 1402, 1377, 1348 cm⁻¹.

Reference Example 13

Ethyl5-(4-aminophenyl)-4-(N-benzyl-N-methylaminomethyl)-2-[N-(2,6-difluorobenzyl)-N-ethoxycarbonylamino]thiophene-3-carboxylate

To a solution of the compound obtained in Reference Example 12 (10.0 g,16.03 mmol) in formic acid (100 ml) were added 1M HCl solution in ether(48 ml, 48 mmol) and 10% palladium-on-carbon (1000 mg) with ice-cooling,and hydrogenation was carried out under atmospheric conditions at roomtemperature for 5 hours. This reaction mixture was filtered with the aidof Celite and the filtrate was concentrated under reduced pressure. Theresidue was partitioned between dichloromethane and saturated aqueoussodium hydrogencarbonate solution, and the aqueous layer was extractedwith dichloromethane. The organic extracts were combined and dried(MgSO₄) and the solvent was distilled off under reduced pressure. Theresidue was chromatographed on silica gel to give the title compound aswhite amorphous powder (7.9 g, 83%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.15-1.31 (6H, m), 1.90 (3H, s), 3.21 (2H,s), 3.65 (2H, s), 3.79 (2H, s), 4.09-4.24 (4H, m), 5.01 (2H, s),6.67-6.80 (4H, m), 7.12-7.26 (8H, m). IR (KBr): 1717, 1628, 1493, 1406,1379 cm⁻¹.

Reference Example 14

Ethyl4-(N-benzyl-N-methylaminomethyl)-2-[N-(2,6-difluorobenzyl)-N-ethoxycarbonylamino]-5-[4-(3-methoxyureido)phenyl]thiophene-3-carboxylate

To a solution of the compound obtained in Reference Example 13 (0.9 g.1.52 mmol) in dichloromethane (20 ml) was added triethylamine (0.43 ml,3.09 mnmol) with ice-cooling. To this solution was addedN,N′-carbonyldiimidazole (0.492 g, 3.03 mmol) with ice-cooling, and themixture was allowed to warm to room temperature and stirred for 48hours. The reaction mixture was ice-cooled again andO-methylhydroxylamine hydrochloride (1.27 g, 15.2 mmol), triethylamine(2.2 ml, 15.8 mmol)and dichloromethane (5 ml) were added. This reactionmixture was allowed to warm to room temperature and stirred for 3 hours.This reaction mixture was partitioned between chloroform and aqueoussodium hydrogencarbonate solution and the aqueous layer was extractedwith chloroform. The organic extracts were combined, washed with aqueoussodium chloride solution and dried (MgSO₄), and the solvent wasdistilled off under reduced pressure. The residue was chromatographed onsilica gel to give the title compound as light-yellow amorphous powder(0.93 g, 92%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.16 (3H, br s), 1.29 (3H, t, J=7.1 Hz), 1.91(3H, s), 3.22 (2H, s), 3.67(2H, s), 3.82(3H, s), 4.17(2H, brs), 4.21(2H,d, J=7.1 Hz), 5.02 (2H, s), 6.78 (2H, t, J=7.8 Hz), 7.12-7.32 (6H, m),7.40 (2H, d, J=8.6 Hz), 7.53 (2H, d, J=8.6 Hz), 7.62(1H, s). IR (KBr):3300, 2982, 1719, 1628, 1591, 1528, 1473, 1408 cm⁻¹.

Reference Example 15

4-(N-Benzyl-N-methylaminomethyl)-2-[N-(2,6-difluorobenzyl)-N-ethoxycarbonylamino]-5-[4-(3-methoxyureido)phenyl]thiophene-3-carboxylicacid

To a solution of the compound obtained in Reference Example 14 (0.1 g,0.15 mmol) in ethanol (2.5 ml) was added a solution of 2N-sodiumhydroxide in water (0.37 ml, 0.74 mmol). This reaction mixture wasstirred at room temperature for 1 hour and at 55° C. for a further 18hours. After cooling, the reaction mixture was neutralized with2N-hydrochloric acid and partitioned between ethyl acetate and saturatedaqueous sodium hydrogencarbonate solution. The aqueous layer wasextracted with ethyl acetate. The organic extracts were combined, washedwith aqueous sodium chloride solution and dried (MgSO₄) and the solventwas distilled off under reduced pressure. The residue waschromatographedon silica gel to give the title compound as colorlessamorphous powder (0.078 g, 81%).

Elemental analysis for C₃₂H₃₂N₄O₆SF₂; C(%) H(%) N(%); Calculated 60.18;5.05; 8.77; Found: 60.00; 5.18; 8.83¹H-NMR (200 MHz, CDCl₃) δ: 1.0-1.35(3H, br s), 2.16 (3H, s), 3.84 (3H, s), 3.84 (2H, s), 3.88 (2H, s),4.10-4.30 (2H, br s), 6.77 (2H, t), 6.70-6.85 (1H, br s), 7.15-7.35 (8H,m), 7.58 (2H, d, J=8.0 Hz), 7.50-7.65 (1H, br s), 7.90-8.00 (1H, br s).

Reference Example 16

4-(N-Benzyl-N-methylaminomethyl)-2-[N-(2,6-difluorobenzyl)-N-ethoxycarbonylamino]-3-(4-methoxymethoxyphenylaminocarbonyl)-5-[4-(3-methoxyureido)phenyl]thiophene

To a solution of the compound obtained in Reference Example 15 (0.80 g,1.23 mmol), triethylamine (0.88 ml, 6.31 mmol) and4-methoxymethoxyaniline (0.96 g, 6.27 mmol) in dichloromethane (25 ml)was added benzotriazol-1-yloxytripyrrolidinophosphoniumhexafluorophosphate (PyBOP) (0.72 g, 1.338 mmol) with ice-cooling. Themixture was allowed to warm to room temperature and stirred for 14hours. This reaction mixture was partitioned between chloroform andsaturated aqueous sodium hydrogencarbonate solution and the aqueouslayer was extracted with chloroform. The organic extracts were combined,washed with aqueous sodium chloride solution and dried (MgSO₄) and thesolvent was distilled off under reduced pressure. The residue waschromatographed on silica gel to give the title compound as light-yellowamorphous powder (0.82 g, 93%).

¹H-NMR (300 MHz, CDCl₃) δ: 1.21 (3H, br s), 2.07 (3H, br s), 3.20 (2H,s), 3.47 (3H, s), 3.68 (2H, s), 3.83 (3H, s), 4.24 (2H, br s), 5.07 (2H,br s), 5.13 (2H, s), 6.75 (2H, t, J=7.9 Hz), 6.93 (2H, d, J=9.0 Hz),7.12-7.18 (3H, m), 7.23-7.25 (4H, m), 7.43 (2H, d, J=9.0 Hz), 7.54 (2H,d, J=8.5 Hz), 7.65 (1H, s). IR (KBr): 3288, 2940, 1717, 1672, 1628,1598, 1564, 1528, 1510, 1473 cm⁻¹.

Reference Example 17

Ethyl2-[N-(2,6-difluorobenzyl)-N-ethoxycarbonylamino]-4-[N-(2-methoxyethyl)-N-methylaminiomethyl]-5-(4-nitrophenyl)thiophene-3-carboxylate

To a solution of the compound obtained in Reference Example 11 (12.82 g,22.0 mmol) ethyl diisopropylamine (7.7 ml, 44.2 mmol) andN-(2-methoxyethyl)methylamine (3.5 ml, 32.6 mmol) in ethyl acetate (120ml) was added. The mixture was stirred for 20 hours at room temperature.This reaction mixture was partitioned between ethyl acetate andsaturated sodium hydrogencarbonate solution and the aqueous layer wasextracted with ethyl acetate. The organic extracts were combined, washedwith aqueous sodium chloride solution and dried (MgSO₄) and the solventwas distilled off under reduced pressure. The residue waschromatographed on silica gel to give the title compound as a brown oil(10.27 g, 79%).

¹H-NMR (300 MHz, CDCl₃) [Free amine] δ: 1.16-1.38 (6H, m), 2.08 (3H, s),2.46 (2H, t, J=6.0 Hz), 3.28 (3H, s), 3.36 (2H, t, J=6.0 Hz), 3.63 (2H,s), 4.09-4.32 (4H, m), 5.01 (2H, s), 6.86 (2H, t, J=8.1 Hz), 7.21-7.32(1H, m), 7.70 (2H, d, J=8.7 Hz), 8.23 (2H, d, J=8.7 Hz). IR (KBr): 2984,1725, 1628, 1597, 1520, 1473 cm⁻¹. FAB-Mass m/z 592(MH)⁺.

Reference Example 18

1-(2,6-Difluorobenzyl)-5-[N-(2-methoxyethyl)-N-methylaminomethyl]-3-(3,4-methylenedioxyphenyl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of 3,4-methylenedioxyaniline (3.30 g, 24.3 mmol) intoluene (80 ml) was added a solution of 1.01 M dimethylaluminiumchloride in hexane (22.2 ml, 22.0 mmol) with ice-cooling. This mixturewas stirred for 1 hour, with ice-cooling. To this reaction mixture wasadded a solution of the compound obtained in Reference Example 17 (2.20g, 3.70 mmol) in toluene (30 ml), and the mixture was stirred for 20hours at room temperature. This reaction mixture was poured intoice-water, and partitioned between ethyl acetate and saturated sodiumhydrogencarbonate solution. The aqueous layer was extracted with ethylacetate. The organic extracts were combined, washed with aqueous sodiumchloride solution and dried (Na₂SO₄) and the solvent was distilled offunder reduced pressure. The residue was chromatographed on silica gel togive the crude product, which was recrystallized from ethylacetate-hexane to give the title compound as brown crystals (0.60 g,68%).

mp: 190-192° C.; Elemental analysis for C₃₁H₂₆N₄O₇SF₂; C(%) H(%) N(%);Calculated: 58.49; 4.12; 8.80; Found 58.50; 3.91; 8.61; ¹H-NMR (300 MHz,CDCl₃) [Free amine] δ: 2.21 (3H, s), 2.68 (2H, t, J=5.7 Hz), 3.31 (3H,s), 3.44 (2H, t, J=5.7 Hz), 3.87 (2H, s), 5.38 (2H, s), 6.03 (2H, s),6.73-6.76 (2H, m), 6.90-6.97 (3H, m), 7.28-7.38 (1H, m), 8.00 (2H, d,J=8.7 Hz), 8.26 (2H, d, J=8.7 Hz). IR (KBr): 2894, 1719, 1671, 1628,1597, 1547, 1520, 1487, 1462, 1348, 1243 cm⁻¹.

Reference Example 19

6-(4-Aminophenyl)-1-(2,6-difluorobenzyl)-5-[N-(2-methoxyethyl)-N-methylaminoethyl]-3-(3,4-methylenedioxyphenyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of the compound obtained in Reference Example 18 (1.56 g,2.50 mmol) in formic acid (30 ml) were added a solution of 1M hydrogenchloride in ether (7.4 ml, 7.4 mmol) and 10% palladium-on-carbon (200mg) with ice-cooling, and hydrogenation was carried out underatmospheric conditions at room temperature with constant stirring for 2hours. This reaction mixture was filtered through Celite and thefiltrate was concentrated under reduced pressure. The residue waspartitioned between chloroform and saturated aqueous sodiumhydrogencarbonate solution. The aqueous layer was extracted withchloroform and the organic extracts were combined, washed with aqueoussodium chloride solution and dried (Na₂SO₄). The solvent was thendistilled off under reduced pressure. The residue was chromatographed onsilica gel to give the crude product, which was recrystallized fromethyl acetate-hexane to give the title compound as brown crystals (1.46g, 96%).

mp: 200-202° C.; Elemental analysis for C₃₁H₂₈N₄O₅SF₂.1.0H₂O; C(%) H(%)N(%); Calculated 59.61; 4.84; 8.97; Found: 59.27; 4.53; 8.48; ¹H-NMR(300 MHz, CDCl₃) [Free amine] δ: 2.13 (3H, s), 2.63 (2H, t, J=5.7 Hz),3.26 (3H, s), 3.41 (2H, t, J=5.7 Hz), 3.80 (2H, s), 5.34 (2H, s), 6.01(2H, s), 6.68-6.76 (4H, m), 6.89-6.93 (3H, m), 7.24-7.39 (3H, m). IR(KBr): 2926, 1715, 1667, 1628, 1533, 1506, 1464 cm⁻¹.

Reference Example 20

5-Chloromethyl-1-(2,6-difluorobenzyl)-3-(3,4-ethylenedioxyphenyl)-6-[4-(3-methoxyureido)phenyl]thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

Using the compound obtained in the following Example 8 as a startingmaterial, the title compound was obtained in the same manner as inReference Example 7.

Yield: 63%; mp: 204-209° C.

Example 1

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione(Example Compound No. 1)

To a solution of the compound obtained in Reference Example 6 (5.0 g,8.41 mmol) in dichloromethane (120 ml) was added triethylamine (2.34 ml,16.82 mmol) with ice-cooling, followed by stirring. To this reactionmixture was added N,N′-carbonyldiimidazole (2.73 g, 16.82 mmol) withice-cooling. The reaction mixture was allowed to warm to roomtemperature and was stirred for 42 hours. The mixture was thenice-cooled again and O-methylhydroxylamine hydrochloride (7.02 g, 84.08mmol) and triethylamine (11.7 ml, 84.08 mmol) were added. The reactionmixture was allowed to warm to room temperature and stirred for 3 hours.This reaction mixture was then partitioned between chloroform andsaturated sodium hydrogencarbonate solution. The aqueous layer wasextracted with chloroform. The extracts were combined, washed withaqueous sodium chloride solution and dried (MgSO₄) and the solvent wasdistilled off under reduced pressure. The residue was chromatographed onsilica gel to give the light-yellow solid, which was recrystallized fromchloroform-ether to give the title compound as white crystals (4.52 g,80%).

mp: 204-205° C.; Elemental analysis for C₃₆H₃₁N₅O₄SF₂; C(%) H(%) N(%);Calculated: 64.75; 4.68; 10.49; Found 64.61; 4.67; 10.31; ¹H-NMR (300MHz, CDCl₃) δ: 2.05(3H, s), 3.57(2H, s), 3.82(3H, s), 3.90(2H, s),5.37(21, s), 6.92(2H, d, J=8.2 Hz), 7.16-7.31(9H, m), 7.42-7.57(5H, m),7.63(1H, s), 7.73(2H, d, J=8.8 Hz). IR (KBr): 3338, 3064, 1717, 1669,1628, 1591, 1531, 1470 cm⁻¹.

Example 2

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dionehydrochloride (Example Compound No. 2)

To a solution of the white crystals obtained in Example 1 (38.34 g,57.42 mmol) in dichloromethane (800 ml) was added hydrogen chloride (1Msolution in diethyl ether) (100 ml) with ice-cooling, and the mixturewas stirred at the same temperature for 10 minutes. This reactionmixture was concentrated under reduced pressure and the residue wasrecrystallized from methanol-ether to give the title compound as whitepowder (40.0 g, 99%).

mp: 182-185° C.; Elemental analysis for C₃₆H₃₁N₅O₄SF₂.HCl.0.5H₂O; C(%)H(%) N(%); Calculated 60.63; 4.66; 9.82; Found: 60.45; 4.68; 9.62; IR(KBr): 3440, 3042, 1713, 1665, 1628, 1593, 1539, 1473 cm⁻¹. FAB-Mass m/z668(MH)⁺.

Example 3

Using the compound obtained in Reference Example 6 as a startingmaterial, Example Compounds No. 3-1 to 3-9 were obtained in the samemanner as in Examples 1 and 2.

Example Compound No. 3-1:

Yield: 91%; mp: 175-180° C. [Hydrochloride].

Example Compound No. 3-2:

Yield: 81%; mp:179-182° C. [Hydrochloride].

Example Compound No. 3-3:

Yield: 80%; mp: 172-177° C. [Hydrochloride].

Example Compound No. 3-4:

Yield: 99%; mp: 193-197° C. [Hydrochloride].

Example Compound No. 3-5:

Yield: 91%; mp: 201-204° C. [Hydrochloride].

Example Compound No. 3-6:

Yield: 89%; mp: 210-215° C. [Hydrochloride].

Example Compound No. 3-7:

Yield: 89%; mp: 199-200° C. [Free amine].

Example Compound No. 3-8:

Yield: 93%; mp: 195-198° C. [Hydrochloride].

Example Compound No. 3-9:

Yield: 95%; mp: 165-170° C. [Hydrochloride].

Example 4

Using the compound obtained in Reference Example 7 as a startingmaterial, Example Compounds No. 4-1 to 4-5 were obtained in the samemanner as in Reference Example 5.

Example Compound No. 4-1:

Yield: 80%; mp: 177-180° C. [Hydrochloride].

Example Compound No. 4-2:

Yield: 77%; mp: 205-210° C. [Hydrochloride].

Example Compound No. 4-3:

Yield: 77%; mp: 182-185° C. [Hydrochloride].

Example Compound No. 4-4:

Yield: 14%; mp: 270° C. (dec) [Hydrochloride].

Example Compound No. 4-5:

Yield: 26%; mp: 260° C. (dec) [Free amine].

Example 5

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-hydroxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of the compound obtained in Reference Example 6 (2.0 g,3.36 mmol) in dichloromethane (40 ml) was added triethylamine (0.94 ml,6.73 mmol) with ice-cooling, followed by stirring. Then,N,N′-carbonyldiimidazole (1.09 g, 6.73 mmol) was added to the reactionmixture with ice-cooling. The reaction mixture was allowed to warm toroom temperature and was stirred for 24 hours. The reaction mixture wasice-cooled again and O-(2,4-dimethoxybenzyl)hydroxylamine (3.11 g, 16.98mmol) was added. The reaction mixture was then allowed to return to roomtemperature and stirred for 19 hours. This reaction mixture waspartitioned between chloroform and saturated aqueous sodiumhydrogencarbonate solution and the aqueous layer was extracted withchloroform. The extracts were combined, washed with aqueous sodiumchloride solution and dried (MgSO₄) and the solvent was distilled offunder reduced pressure. To a solution of the residue in dichloromethane(50 ml) was added trifluoroacetic acid (5 ml), followed by stirring atroom temperature for 20 minutes. This reaction mixture was partitionedbetween chloroform and saturated aqueous sodium hydrogencarbonatesolution, and the aqueous layer was extracted with chloroform. Theextracts were combined, washed with aqueous sodium chloride solution anddried (MgSO₄) and the solvent was distilled off under reduced pressure.The residue was chromatographed on silica gel to give white amorphouspowder, which was recrystallized from chloroform-ether to give the titlecompound as white crystals (2.2 g, 100%).

mp: 164-165° C.; ¹H-NMR (300 MHz, CDCl₃) δ: 2.05 (3H, s), 3.46 (2H, s),3.92 (2H, s), 5.35 (2H, s), 6.65(1H, br), 6.90 (2H, t, J=8.0 Hz),7.28-7.65 (15H, m), 8.04 (1H, s), 9.73 (1H, br). IR (KBr): 3326, 2856,1715, 1665, 1628, 1591, 1531, 1468 cm⁻¹. FAB-Mass m/z 654(MH)⁺.

Example 6

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-hydroxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dionehydrochloride

In a solution of the white crystals obtained in Example 5 (60 mg, 0.094mmol) in dichloromethane (5 ml) was added hydrogen chloride (1M solutionin diethyl ether) (0.2 ml) with ice-cooling, followed by stirring at thesame temperature for 10 minutes. This reaction mixture was concentratedunder reduced pressure and the residue was recrystallized frommethanol-ether to give the title compound as white powder (72 mg, 100%).

mp: 180-186° C.; Elemental analysis for C₃₅H₂₉N₅O₄SF₂.0.1HCl.1.0H₂O;C(%) H(%) N(%); Calculated: 59.36; 4.55: 9.89; Found: 59.37; 4.60; 9.87;IR(KBr): 3388, 3066, 1713, 1663, 1628, 1593, 1537, 1473cm⁻¹.

Example 7

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-[3-(2-hydroxyethyl)ureido]phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of the Example Compound No. 3-7 (900 mg, 1.24 mmol) in THF(20 ml) was added 5N-potassium hydroxide solution in water (7 ml) withice-cooling, followed by stirring at 60° C. for 1 hour. This reactionmixture was partitioned between ethyl acetate and saturated sodiumchloride solution and the aqueous layer was extracted with ethylacetate. The extracts were combined, washed with aqueous sodium chloridesolution and dried (MgSO₄) and the solvent was distilled off underreduced pressure. The residue was chromatographed on silica gel to givethe white amorphous powder, which was recrystallized fromchloroform-methanol-ether to give the title compound as white crystals(850 mg, 88%).

mp: 220-222° C.; Elemental analysis for C₃₇H₃₃N₅O₄SF₂; C(%) H(%) N(%);Calculated: 65.18; 4.88; 10.27; Found: 65.08; 5.01; 10.29; ¹H-NMR (300MHz, DMSO-d₆) δ: 1.93 (3H, s), 3.17 (2H, q, J=4.8 Hz), 3.45-3.47 (4H,m), 3.81 (2H, s), 4.76 (1H, t, J=5.1 Hz), 5.28 (2H, s), 6.28(1H, t,J=5.4 Hz), 7.12-7.28 (9H, m), 7.44-7.58 (8H, m), 8.79 (lH, s). IR(KBr):3530, 3364, 3066, 2958, 2884, 1715, 1667, 1595, 1531, 1470 cm⁻¹.FAB-Mass m/z 682(MH)⁺.

Example 8

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-3-(3,4-ethylenedioxyphenyl)-6-[4-(3-methoxyureido)phenyl]thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

To a solution of 3,4-ethylenedioxyaniline (3.90 g, 25.8 mmol) indichloromethane (100 ml) was added a solution of 1.01M dimethylaluminumchloride in hexane (25.5 ml, 25.8 mmol) under ice-cooling. The mixturewas allowed to warm to room temperature with stirring for 1 hour. Tothis solution was added a solution of the compound obtained in ReferenceExample 14 (3.44 g, 5.16 mmol) in dichloromethane (60 ml), and themixture was stirred at room temperature for 1 day. This reaction mixturewas partitioned between chloroform and saturated aqueous sodium chloridesolution and the aqueous layer was extracted with chloroform. Theorganic layers were combined, washed with aqueous sodium chloridesolution and dried (MgSO₄) and the solvent was distilled off underreduced pressure. The residue was chromatographed on silica gel to givethe title compound as white amorphous powder (3.2 g, 85%).

mp: 185-187° C.; Elemental analysis for C₃₈H₃₄N₅O₆SF₂Cl.H₂O; C(%) H(%)N(%); Calculated: 58.50; 4.65; 8.98; Found: 58.73; 4.48; 9.07; ¹H-NMR(300 MHz, CDCl₃) δ: 2.05 (3H, s), 3.57 (2H, s), 3.83 (3H, s), 3.90 (2H,s), 4.29 (4H, s), 5.35 (2H, s), 6.75-7.01 (5H, m), 7.12-7.33 (7H, m),7.55 (2H, d, J=8.0 Hz), 7.63 (1H, s), 7.72 (2H, d, J=8.0 Hz). IR(KBr):1717, 1702, 1686, 1657, 1636, 1626, 1560, 1543, 1522, 1510, 1475 cm⁻¹.

Example 9

Using the compound obtained in Reference Example 14 as a startingmaterial, Example Compounds No. 9-1 to 9-2 were obtained in the samemanner as in Example 8.

Example Compound No. 9-1:

Yield: 60%; mp: 148-151° C. [Free amine].

Example Compound No. 9-2:

Yield: 54%; mp: 169-170° C. [Hydrochloride].

Example 10

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-3-[4-(methoxymethoxy)phenyl]-6-[4-(3-methoxyureido)phenyl]thieno[2,3-d]pyrimidine-2,4-(1H,3H)-dione(Example Compound No. 10)

To a solution of the compound obtained in Reference Example 16 (0.84 g,1.09 mmol) in anhydrous methanol (50 ml) was added a solution of sodiummethoxide (2.10 g, 10.4 mmol) in anhydrous methanol (20 ml) withice-cooling. This mixture was stirred for 2.5 hours, while thetemperature was allowed to warm to room temperature. This reactionmixture was neutralized with 1N-hydrochloric acid (10.9 ml, 10.9 mmol)and the solvent was distilled off under reduced pressure. The residuewas partitioned between chloroform and saturated aqueous sodium chloridesolution and the aqueous layer was extracted with chloroform. Theorganic extracts were combined, washed with aqueous sodium chloridesolution and dried (MgSO₄) and the solvent was distilled off underreduced pressure. The residue was recrystallized from ethylacetate-isopropyl ether to give the title compound as white crystals(0.632 g, 80%).

mp: 189-191° C.;

Elemental analysis for C₃₈H₃₅N₅O₆SF₂; C(%) H(%) N(%); Calculated 62.71;4.85; 9.62; Found 62.56; 4.69; 9.33; ¹H-NMR (300 MHz, CDCl₃) δ: 2.05(3H, s), 3.49 (3H, s), 3.57 (2H, s), 3.82 (3H, s), 3.91 (2H, s), 5.21(2H, s), 5.36 (2H, s), 6.92 (2H, d, J=8.0 Hz), 7.14-7.35 (11H, m), 7.55(2H, d, J=8.5 Hz), 7.63 (1H, s), 7.72 (2H, d, J=8.5 Hz). IR (KBr): 3380,2940, 2830, 1717, 1703, 1669, 1628, 1589, 1524, 1464 cm⁻¹.

Example 11

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-3-(4-hydroxyphenyl)-6-[4-(3-methoxyureido)phenyl]thieno[2,3-d]pyrimidine-2,4-(1H,3H)-dione

The Example Compound No. 10 (0.35 g, 0.48 mmol) was dissolved in acetone(10 ml). and then was added 6N-hydrochloric acid (1.0 ml, 6.0 mmol). Themixture was stirred at room temperature for 6 hours and neutralized witha solution of 2N-sodium hydroxide (3 ml, 6.0 mmol) in water withice-cooling and the solvent was distilled off under reduced pressure.The residue was partitioned between chloroform and saturated aqueoussodium chloride solution and the aqueous layer was extracted withchloroform. The organic extracts were combined, washed with aqueoussodium chloride solution and dried (MgSO₄) and the solvent was distilledoff under reduced pressure. The residue was chromatographed on silicagel to give colorless amorphous powder (0.18 g, 55%), which wasrecrystallized from chloroform-methanol to give the title compound aswhite crystals (0.067 g).

mp: 178-182° C.; Elemental analysis for C₃₆H₃₁N₅O₅SF₂.0.4H2O; C(%) H(%)N(%); Calculated 62.58; 4.64; 10.14; Found: 62.78; 4.57; 9.86; ¹H-NMR(300 MHz, CDCl₃) δ: 2.04 (3H, s), 3.56 (2H, s), 3.80 (3H, s), 3.90 (2H,s), 5.35 (2H, s), 6.89-6.98 (4H, m), 7.08 (2H, d, J=8.8 Hz), 7.15-7.31(6H, m), 7.57 (2H, d, J=8.6 Hz), 7.69(2H, d, J=8.6 Hz), 7.87(1H, s),8.27(1H, s), 8.88 (1H, s). IR (KBr): 3446, 1717, 1663, 1630, 1601, 1534,1520, 1473 cm⁻¹.

Example 12

5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-14-[(3-methoxy-3-methoxycarbonyl)ureido]phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4-(1H,3H)-dione

To a solution of the Example Compound No. 1 (0.334 g, 0.5 mmol) intetrahydrofuran (10 ml) were added triethylamine (0.08 ml, 0.6 mmol) andmethyl chloroformate (0.0425 ml, 0.55 mmol) with ice-cooling and themixture was stirred for 1 hour with ice-cooling and then for 1 hour atroom temperature. To this mixture were added triethylamine (0.08 ml, 0.6mmol) and ethyl chloroformate (0.0425 ml, 0.55 mmol), and the mixturewas stirred for 2 hours at 40° C. and then for 2 hours at roomtemperature. To this mixture was added aqueous sodium chloride solutionand extracted with ethyl acetate. The extracts were combined, washedwith aqueous sodium chloride solution and dried (MgSO₄) and the solventwas distilled off under reduced pressure. The residue waschromatographed on silica gel to give the crude product, which wasrecrystallized from ethyl acetate-diethyl ether to give the titlecompound as colorless crystals (0.204 g, 56%).

mp: 150-152° C.; Elemental analysis for C₃₈H₃₃N₅O₆SF₂; C(%) H(%) N(%);Calculated: 62.89; 4.58: 9.65; Found 62.68; 4.69; 9.44; ¹H-NMR (200 MHz,CDCl₃) δ: 2.06 (3H, s), 3.57 (2H, s), 3.91 (2H, s), 3.93 (3H, s), 3.98(3H, s), 5.37 (2H, s), 6.92 (2H, t, J=8.2 Hz), 7.15-7.60 (11H, m), 7.57(2H, d, J=8.6 Hz), 7.73 (2H, d, J=8.6 Hz), 10.06 (1H, s). IR (KBr):1746, 1713, 1663, 1537, 1460, 1339, 1200, 1034, 737 cm⁻¹.

Example 13

1-(2,6-Difluorobenzyl)-5-[N-(2-methoxyethyl)-N-methylaminomethyl]-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4-(1H,3H)-dione

To a solution of the compound obtained in Reference Example 7 (0.86 g,1.48 mmol) in N,N-dimethylformamide (15 ml) were addedethyldiisopropylamine (0.34 ml, 1.92 mmol), potassium iodide (245 mg,1.48 mmol) and N-(2-methoxyethyl)methylamine (0.19 ml, 1.78 mmol), andthe mixture was stirred at room temperature for 2 hours. This reactionmixture was concentrated to give the residue, which was partitionedbetween ethyl acetate and saturated aqueous sodium hydrogencarbonatesolution. The aqueous layer was extracted with ethyl acetate. Thecombined extracts were washed with aqueous sodium chloride solution anddried (MgSO₄) and the solvent was distilled off under reduced pressure.The residue was chromatographed on silica gel to give the title compoundas white crystals (840 mg, 89%).

mp: 161-163° C.; Elemental analysis for C₃₂H₃₁N₅O₅SF₂.0.5H₂O; C(%) H(%)N(%); Calculated 59;62; 5.00; 10.86; Found 59.73; 4.99: 10.85; ¹H-NMR(300 MHz, CDCl₃) [Free amine] δ: 2.14 (3H, s), 2.64 (2H, t, J=5.9 Hz),3.27 (3H, s), 3.41 (2H, t, J=5.9 Hz), 3.83 (5H, s), 5.37 (2H, s), 6.93(2H, t, J=8.2 Hz), 7.12-7.63 (12H, m). IR (KBr): 1709, 1663, 1560, 1522cm⁻¹.

Example 14

1-(2,6-Difluorobenzyl)-3-(3,4-ethylenedioxyphenyl)-5-[N-(2-methoxyethyl)-N-methylaminomethyl]-6-[4-(3-methoxyureido)phenyl]thieno[2,3-d]pyrimidine-2,4-(1H,3H)-dione

Using the compounds obtained in Reference Example 20 as startingmaterial, the title compound was obtained in the same manner as inExample 13.

Yield: 79%; mp: 155-156° C. [Free amine].

Example 15

1-(2,6-Difluorobenzyl)-5-[N-(2-methoxyethyl)-N-methylaminomethyl]-6-[4-(3-methoxyureido)phenyl]-3-(3,4-methylenedioxyphenyl)thieno[2,3-d]pyrimidine-2,4-(1H,3H)-dione

Using the compound obtained in Reference Example 19 as a startingmaterial, the title compound was obtained in the same manner as inExample 1.

Yield: 72%; mp: 150-152° C. [Free amine].

Preparation Example 1

Using 100 mg of the Example Compound No. 1, 165 mg of lactose, 25 mg ofcorn starch, 4 mg of polyvinyl alcohol and 1 mg of magnesiumstearate,.tablets are produced by a conventional method.

Preparation Example 2

The Example Compound No. 2 (5 g) is dissolved in distilled water forinjection to make a total volume of 100 ml. This solution is asepticallyfiltered through a 0.22 μm membrane filter (produced by SumitomoElectric Industries, Ltd. or Sartorius) and dispensed at 2 ml per washedsterile vial, followed by freeze-drying by a conventional method, toyield a 100 mg/vial freeze-dried injectable preparation.

Preparation Example 3

Using 100 mg of the Example Compound No. 4-2, 165 mg of lactose, 25 mgof corn starch, 4 mg of polyvinyl alcohol and 1 mg of magnesiumstearate, tablets are produced by a conventional method.

Preparation Example 4

The Example Compound No. 4-2 (5 g) is dissolved in distilled water forinjection to make a total volume of 100 ml. This solution is asepticallyfiltered through a 0.22 μm membrane filter (produced by SumitomoElectric Industries, Ltd. or Sartorius) and dispensed at 2 ml per washedsterile vial, followed by freeze-drying by a conventional method, toyield a 100 mg/vial freeze-dried injectable preparation.

Preparation Example 5

(1) Example Compound No. 1 or No. 4-2 5 g (2) Lactose/crystallinecellulose (particles) 330 g (3) D-mannitol 29 g (4) Low-substitutionalhydroxypropyl cellulose 20 g (5) Talc 25 g (6) Hydroxypropyl cellulose50 g (7) Aspartame 3 g (8) Dipotassium glycyrrhizinate 3 g (9)Hydroxypropylmethyl cellulose 2910 30 g (10)  Titanium oxide 3.5 g (11) Yellow iron sesquioxide 0.5 g (12)  Light silicic anhydride 1 g

Components (1), (3), (4), (5), (6), (7) and (8) are suspended ordissolved in purified water and coated on the core particles (2) toyield base fine subtilae, which are then further coated with components(9) through (11) to yield coated fine subtilae, which are then mixedwith component (12) to yield 500 g of 1% fine subtilae of the compound.These subtilae are divided to 500 mg folded subtilae.

Experimental Example 1

(1) Preparation of ¹²⁵I-leuprorelin

To a tube containing 10 μl of a 3×10⁻⁴ M aqueous solution of leuprorelinand 10 μl of 0.01 mg/ml lactoperoxidase, 10 gl (37 MBq) of a solution ofNa¹²⁵I was added. After stirring, 10 μl of 0.001% H₂O₂ was added, and areaction was carried out at room temperature for 20 minutes. By adding700 μl of a 0.05% TFA (trifluoroacetic acid) solution, the reaction wasstopped, followed by purification by reversed-phase HPLC. The HPLCconditions used are shown below. ¹²⁵I-leuprorelin was eluted at aretention time of 26 to 27 minutes

Column: TSKgel ODS-80™ (TM indicates a registered trademark; the sameapplies below) CTR (4.6 mm×10 cm)

Eluents: Solvent A (0.05% TFA)

Solvent B (40% CH₃CN−0.05% TFA)

0 minute (100% Solvent A)—3 minutes (100% Solvent A)—7 minutes (50%Solvent A+50% Solvent B)—40 minutes (100% Solvent B)

Eluting temperature: Room temperature

Elution rate: 1 ml/min

(2) Preparation of a Rat Pituitary Anterior Lobe Membrane FractionContaining GnRH Receptors

Anterior lobes of the pituitary glands were isolated from forty Wistarrats (8 weeks old, male), and washed with ice-cooled homogenate buffer[25 mM Tris (tris(hydroxymethyl)aminomethane)-HCl, 0.3 M sucrose, 1 mMEGTA (glycol-etherdiamine-N,N,N′,N′-tetraacetic acid), 0.25 mM PMSF(phenylmethylsulfonyl fluoride), 10 U/ml aprotinin, 1 μg/ml pepstatin,20 μg/ml leupeptin, 100 μg/ml phosphoramidon, 0.03% sodium azide, pH7.5]. The pituitary tissue was floated in 2 ml of the homogenate bufferand homogenized using a Polytron homogenizer. The homogenate wascentrifuged at 700×g for 15 minutes. The supernatant was taken in anultracentrifuge tube and centrifuged at 100,000×g for 1 hour to give amembrane fraction pellet. This pellet was suspended in 2 ml of assaybuffer [25 mM Tris-HCl, 1 mM EDTA (ethylenediaminetetraacetic acid),0.1% BSA (bovine serum albumin), 0.25 mM PMSF, 1 μg/ml pepstatin, 20μg/ml leupeptin, 100 μg/ml phosphoramidon, 0.03% sodium azide, pH 7.5]and the suspension was centrifuged at 100,000×g for 1 hour. The membranefraction recovered as a pellet was resuspended in 10 ml of assay buffer,divided into portions, preserved at −80° C. and thawed when needed.

(3) Preparation of CHO (Chinese hamster ovarian) Cell Membrane FractionContaining Human GnRH Receptor

Human GnRH receptor-expressing CHO cells (10⁹ cells) were suspended inphosphate-buffered saline supplemented with 5 mM EDTA(ethylenediaminetetraacetic acid) (PBS-EDTA) and centrifuged at 100×gfor 5 minutes. To the cell pellet, 10 ml of a cell homogenate buffer (10mM NaHCO₃, 5 mM EDTA, pH 7.5) was added, followed by homogenizationusing the Polytron homogenizer. After centrifugation at 400×g for 15minutes, the supernatant was transferred to an ultracentrifugation tubeand centrifuged at 100,000×g for 1 hour to yield a membrane fractionprecipitate. This precipitate was suspended in 2 ml of an assay bufferand centrifuged at 100,000×g for 1 hour. The membrane fraction recoveredas a precipitate was again suspended in 20 ml of the assay buffer,dispensed, and stored at −80° C. before use upon thawing.

(4) Determination of ¹²⁵I-leuprorelin Binding Inhibition Rate

The rat and human membrane fractions prepared in the above (2) and (3)were diluted with the assay buffer to yield a 200 μg/ml dilution, whichwas then dispensed at 188 μl per tube. Where the rat pituitary anteriorlobe membrane fraction was used, to each tube, 2 μl of a solution of 0.1mM compound in 60% DMSO (dimethyl sulf oxide) and 10 μl of 38 nM¹²⁵I-leuprorelin were added simultaneously. Where the cell membranefraction of the CHO with human GnRH receptors expressed, to each tube, 2μl of a solution of 2 mM compound in 60% DMSO and 10 μl of 38 nM¹²⁵I-leuprorelin were added simultaneously. To determine maximum bindingquantity, a reaction mixture of 2 μl of 60% DMSO and 10 μl of 38 nM¹²⁵I-leuprorelin was prepared. To determine non-specific binding amount,a reaction mixture of 2 μl of 100 μM leuprorelin in solution in 60% DMSOand 10 μlof 38 nM ¹²⁵I-leuprorelin was prepared.

Where the rat pituitary anterior lobe membrane fraction was used, thereaction was conducted at 4° C. for 90 minutes. Where the membranefraction of the CHO with human GnRH receptors expressed was used, thereaction was carried out at 25° C. for 60 minutes. After each reaction,the reaction mixture was aspirated and filtered through apolyethyleneimine-treated Whatman glass filter (GF-F). After thisfiltration, the radioactivity of ¹²⁵I-leuprorelin remaining on thefilter paper was measured with a γ-counter.

The expression (TB-SB)/(TB-NSB)×100 (where SB=radioactivity with thecompound added, TB=maximum bound radioactivity, NSB=nonspecificallybound radioactivity) was calculated to find the binding inhibition rate(%) of each test compound. Furthermore, the inhibition rate wasdetermined by varying the concentration of the test substance and the50% inhibitory concentration (IC₅₀ value) of the compound was calculatedfrom Hill plot. The results are shown in below.

binding inhibition I₅₀ value rate (%) (μM) Test Compound Rat (1 μM)Human (20 μM) Rat Human Ex. Compd. No. 2 27 NT NT 0.0001 Ex. Compd. No.4-2 64 NT 0.5 0.0002 NT not measured

Experimental Example 2

Suppression of plasma LH in castrated monkeys

The Example compound No. 2 was orally administered to castrated malecynomolgus monkeys (Macaca fascicularis), and plasma LH was quantified.The male cynomolgus monkeys, used at 4 years 9 months to 6 years 3months of age at time of experimentation, had been castrated more than 3months prior to the examination. Test animals [n=3] were given 30 mg/kg(3 ml/kg) of the compound suspended in 0.5% methyl cellulose at a finalconcentration of 1% by oral administration, and control animals [n=2]were given 3 ml/kg of the 0.5% methyl cellulose dispersant alone by oraladministration. At 24 hours and immediately before administration and at2, 4, 6, 8, 24, and 48 hours after administration, blood was collectedfor heparinized plasma samples via the femoral vein and immediatelystored under freezing conditions.

Plasma LH concentrations were determined by a bioassay using mousetesticular cells. The testicular cells were collected from male BALB/cmice (8 to 9 weeks of age) and washed three times with 1 ml ofDulbecco's modified Eagle medium (DMEM-H) containing 20 mM HEPES and0.2% BSA per testis.

After incubation at 37° C. for 1 hour, the cells were passed through anylon mesh filter (70 μm) and dispensed to test tubes at 8×10⁵cells/tube. After the cells were washed twice with 0.4 ml of DMEM-H, 0.4ml of a DMEM-H solution containing either equine LH (Sigma Company), asthe standard LH, or monkey plasma, previously diluted up to 300 fold, asthe test sample, was added, followed by a reaction at 37° C. for 2hours. The testosterone concentration in the culture supernatant wasdetermined by a radioimmunoassay (CIS Diagnostics Company), and the LHconcentration in the test monkey plasma was calculated from the standardcurve for the standard equine LH.

The results are given together in FIG. 1.

The LH concentration is expressed in the percentage (%) of the baselineLH concentration immediately before administration in each individualcynomolgus monkey and is shown as the time course with theadministration time being taken as 0 (indicated by the arrowmark) andvalues before and after administration being indicated by the minus andplus signs, respectively. The control group-1 (-▴-) and control group-2(-♦-) were orally dosed with 0.5% methylcellulose dispersant (3 ml/kg)only, while the compound group-1 (-Δ-), compound group-2 (-□-) andcompound group-3 (-◯-) similarly received a dispersion of the ExampleCompound No. 2 in 0.5% methylcellulose (30 mg/kg, 3 ml/kg).

The control groups showed little change in the plasma LH concentrationeven after administration. On the other hand, in the compound groups,the plasma LH concentration showed a rapid fall beginning immediatelyafter administration and had fallen to 20% of the baseline or less by 24hours after administration. Then, at 48 hours after administration,re-elevation of the plasma LH concentration was noted.

The above results indicate that the Example Compound No. 2, administeredorally, has a significant depressive effect on blood LH concentration.

It is evident from the foregoing results that compounds of the presentinvention antagonize the pituitary LH-RH receptors to block the LH-RHstimulation from the hypothalamus to inhibit the LH release.

INDUSTRIAL APPLICABILITY

A compound of the present invention possesses excellentgonadotropin-releasing hormone antagonizing activity. It is also good inoral absorbability and excellent in stability and pharmacokinetics. Withlow toxicity, it is also excellent in safety. Therefore, the compound ofthe present invention can be used as a prophylactic or therapeutic agentfor hormone-dependent diseases etc. Concretely, it is effective as aprophylactic or therapeutic agent for sex hormone-dependent cancers(e.g., prostatic cancer, uterine cancer, breast cancer, pituitary tumor,etc.), prostatic hypertrophy, hysteromyoma, endometriosis. precociouspuberty, amenorrhea syndrome, multilocular ovary syndrome, pimples etc,or as a pregnancy regulator (e.g., contraceptive), infertility remedy ormenstruation regulator. It is also effective as an animal estrousregulator, food meat quality improving agent or animal growth regulatorin the field of animal husbandry, and as a fish spawning promoter in thefield of fishery.

What is claimed is: 1.5-(N-Benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dioneor a salt thereof.